U.S. patent application number 09/946914 was filed with the patent office on 2003-03-06 for regenerative bone implants.
Invention is credited to Chan, Wing P., Deng, Wen-Ping, Lai, Wen-Fu T., Pham, Wellington, Tsai, Yu-Hui, Yang, Wei-Chung.
Application Number | 20030045942 09/946914 |
Document ID | / |
Family ID | 25485174 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030045942 |
Kind Code |
A1 |
Lai, Wen-Fu T. ; et
al. |
March 6, 2003 |
Regenerative bone implants
Abstract
The present invention features a regenerative bone implant. The
implant includes a biocompatible and biodegradable matrix having
pores, and a biocompatible and biodegradable biopolymer disposed in
the pores and covalently bonded to the matrix. Optionally, the
implant can further include a bone formation promoter that is also
disposed in the pores and can be covalently bonded to the
matrix.
Inventors: |
Lai, Wen-Fu T.; (Taipei,
TW) ; Deng, Wen-Ping; (Tao-Yuan, TW) ; Tsai,
Yu-Hui; (Yunghe City, TW) ; Chan, Wing P.;
(Taipei, TW) ; Yang, Wei-Chung; (Taipei, TW)
; Pham, Wellington; (Toledo, OH) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
25485174 |
Appl. No.: |
09/946914 |
Filed: |
September 5, 2001 |
Current U.S.
Class: |
623/23.51 ;
523/115; 623/23.58; 623/23.61; 623/901; 623/908 |
Current CPC
Class: |
A61L 27/425 20130101;
A61F 2310/00293 20130101; A61F 2/28 20130101; C08L 89/06 20130101;
A61L 2430/02 20130101; A61F 2210/0004 20130101; A61L 27/227
20130101; A61F 2002/30062 20130101; A61L 27/56 20130101; A61F
2310/00365 20130101 |
Class at
Publication: |
623/23.51 ;
623/23.58; 623/23.61; 623/901; 523/115; 623/908 |
International
Class: |
A61F 002/28; C08L
001/00 |
Claims
What is claimed is:
1. A regenerative bone implant comprising: a biocompatible and
biodegradable matrix having pores, and a biocompatible and
biodegradable biopolymer disposed in the pores and covalently
bonded to the matrix.
2. The regenerative bone implant of claim 1, further comprising a
bone formation promoter that is also disposed in the pores.
3. The regenerative bone implant of claim 2, wherein the bone
formation promoter is covalently bonded to the biocompatible and
biodegradable matrix.
4. The regenerative bone implant of claim 2, wherein the
biocompatible and biodegradable matrix is a hydroxyapatite-based
matrix.
5. The regenerative bone implant of claim 4, wherein the
biocompatible and biodegradable biopolymer is collagen.
6. The regenerative bone implant of claim 5, wherein the bone
formation promoter is osteoprotegerin.
7. The regenerative bone implant of claim 2, wherein the
biocompatible and biodegradable matrix is a hydroxyapatite-based
matrix.
8. The regenerative bone implant of claim 2, wherein the
biocompatible and biodegradable biopolymer is collagen.
9. The regenerative bone implant of claim 1, wherein the
biocompatible and biodegradable matrix is a hydroxyapatite-based
matrix.
10. The regenerative bone implant of claim 9, wherein the
biocompatible and biodegradable biopolymer is collagen.
11. The regenerative bone implant of claim 1, wherein the
biocompatible and biodegradable biopolymer is collagen.
12. A method for preparing a regenerative bone implant, comprising:
providing a biocompatible and biodegradable matrix having pores,
providing a liquid containing a biocompatible and biodegradable
biopolymer, immersing the matrix in the liquid, and thereby
disposing the biopolymer in the pores.
13. The method of claim 12, further comprising covalently bonding
the biopolymer in the pores to the biocompatible and biodegradable
matrix.
14. The method of claim 13, wherein the biocompatible and
biodegradable matrix is a hydroxyapatite-based matrix.
15. The method of claim 14, wherein the biocompatible and
biodegradable biopolymer is collagen.
16. The method of claim 13, wherein the biocompatible and
biodegradable biopolymer is collagen.
17. The method of claim 12, wherein the liquid further contains a
bone formation promoter to be disposed in the pores.
18. The method of claim 17, further comprising covalently bonding
the biocompatible and biodegradable biopolymer in the pores to the
biocompatible and biodegradable matrix.
19. The method of claim 18, further comprising covalently bonding
the bone formation promoter to the biocompatible and biodegradable
matrix in the pores.
20. The method of claim 18, wherein the biocompatible and
biodegradable matrix is a hydroxyapatite-based matrix.
21. The method of claim 20, wherein the biocompatible and
biodegradable biopolymer is collagen.
22. The method of claim 21, wherein the bone formation promoter is
osteoprotegerin.
23. The method of claim 18, wherein the biocompatible and
biodegradable biopolymer is collagen.
24. The method of claim 23, wherein the bone formation promoter is
osteoprotegerin.
25. The method of claim 18, wherein the bone formation promoter is
osteoprotegerin.
26. A method for treating a bone defect in a subject, comprising
replacing the bone defect with a regenerative bone implant, wherein
the implant includes a biocompatible and biodegradable matrix
having pores, and a biocompatible and biodegradable biopolymer
disposed in the pores and covalently bonded to the matrix.
27. The method of claim 21, wherein the regenerative bone implant
further includes a bone formation promoter that is also disposed in
the pores.
28. The method of claim 27, wherein the bone formation promoter is
covalently bonded to the biocompatible and biodegradable
matrix.
29. The method of claim 27, wherein the biocompatible and
biodegradable matrix is a hydroxyapatite-based matrix.
30. The method of claim 28, wherein the biocompatible and
biodegradable biopolymer is collagen.
31. The method of claim 29, wherein the bone formation promoter is
osteoprotegerin.
32. The method of claim 26, wherein the biocompatible and
biodegradable matrix is a hydroxyapatite-based matrix.
33. The method of claim 26, wherein the biocompatible and
biodegradable biopolymer is collagen.
Description
BACKGROUND
[0001] Bone implantation is necessary when a bone fails to repair
itself at a normal rate or when bone loss occurs as a result of
fractures or diseases. A metal implant can be used as an internal
fixation to support the fracture healing, but it has limited use
due to mutagenicity and mechanical properties. See, e.g., Laftman
(1980) Acta Orthop Scand 51(2): 215-22; (1989) supra 60(6): 718-22;
van der List et al. (1988) Acta Orthop Scand 59(3): 328-30; and
Penman et al. (1984) J Bone Joint Surg Br 66(5): 632-4. A bone
implant can also be a graft, such as an autograft, an allograft, or
a xenograft. Use of an autograft, a tissue transplanted from one
site to another in a patient, has the advantage of avoiding immune
responses. However, it requires a second surgery and therefore has
a higher risk of infection. An allograft is a tissue taken from a
different organism of the same species, and a xenograft from an
organism of a different species. Both allografts and xenografts
elicit immune responses.
SUMMARY
[0002] The present invention features a regenerative bone implant
that includes a matrix having pores (including interstices), and a
biopolymer disposed in the pores and covalently bonded to the
matrix. Both the matrix and the biopolymer are biocompatible and
biodegradable. The implant may further include a bone formation
promoter that is also disposed in the pores, and if preferred, is
also covalently bonded to the matrix.
[0003] The term "matrix" herein refers to a material that can be
prepared from an inorganic compound (e.g., hydroxyapatite) or from
an organic polymer (e.g., polylactic acid or polyglyclic acid), and
is capable of mechanical strength in lieu of the bone to be
replaced. The term "biopolymer" herein refers to a protein (e.g.,
collagen) or a protein-containing macromolecule (e.g.,
proteoglycan) that can function as a scaffold for cell attachment
and migration to facilitate regeneration of new bone tissues. The
biopolymer is disposed in the pores of the matrix for more
efficient cell migration and ingrowth. A bone formation promoter is
an agent that promotes growth of bone tissues and maintenance of
bone mass, e.g., osteoprotegerin.
[0004] This invention also features a method for preparing a
regenerative bone implant. The method includes providing a
just-described matrix having pores, providing a liquid containing a
just-described biopolymer, immersing the matrix in the liquid, and
thereby disposing the biopolymer in the pores. It can further
include covalently binding the biopolymer to the matrix. The liquid
may further contain a bone formation promoter to be deposited in
the pores of the matrix. Optionally, the bone formation promoter is
covalently bonded to the matrix.
[0005] Also within the scope of this invention is a method for
treating a bone defect in a subject by replacing the bone defect
with a regenerative bone implant described above.
[0006] Other features or advantages of the present invention will
be apparent from the following detailed description of several
embodiments, and also from the appending claims.
DETAILED DESCRIPTION
[0007] This invention features a regenerative bone implant that is
biocompatible and biodegradable. More specifically, the implant
includes a porous matrix, a biopolymer, and optionally, a bone
formation promoter. The biopolymer is deposited in the pores of the
matrix and covalently bonded to the matrix. The bone formation
promoter, if present, is also deposited in the pores of the matrix,
and may or may not be covalently bonded to the matrix.
[0008] An example of a matrix to be used to prepare an implant of
this invention is a hydroxyapatite-based matrix that includes
hydroxyapatite as the major component. Hydroxyapatite, naturally
occurring in, e.g., bones, enamel, or dentin, has been used for
years as a bone substitute or a coating material. See, for example,
Frame (1987) Int. J. Oral Maxillofacial Surgery 16: 642-55, and
Parsons, et al. (1988) Annals N.Y. Academy of Sciences 523:
190-207. Hydroxyapatite can be prepared by well-known methods or
purchased from commercial suppliers. It is either a pure compound
of Ca.sub.10(PO.sub.4).sub.6(OH).sub.2, or a composition containing
other ions, such as carbonate, fluoride, chloride, or barium. When
using a hydroxyapatite-based matrix to prepare a bone implant of
this invention, the matrix can be hydrothermally processed to
obtain desired pore sizes, such as 150 .mu.m to 350 .mu.m, or 200
.mu.m to 300 .mu.m in diameter. In order to covalently bond a
biopolymer to a hydroxyapatite-based matrix, the surfaces of the
matrix, in particular the interior surfaces of the pores, are first
modified with functional groups, such as amino or hydroxyl. The
functional groups can be introduced by plasma deposition or
chemical priming. Materials used in plasma deposition include, but
are not limited to, ammonia plasma, allylamine plasma, allylalcohol
plasma, and plasma of any gas containing amino, hydroxyl, or other
reactive groups. Compounds used in chemical priming can be amino
silanes, hydroxyl silanes, or other silanes containing amino,
hydroxyl, or other reactive groups. See, e.g., Sano et al. (1993)
Biomaterials 14: 817-822; and Wang and Hsiue (1993) J. Polymer
Science, Part A: Polymer Chemistry 31: 2601-2607.
[0009] An example of a biopolymer to be used to prepare an implant
of this invention is collagen. Collagen, e.g., type I collagen, can
be isolated from human or animal tissues, such as tendon, skin,
bone, or ligament. See, for example, Miller and Rhodes, (1982)
Methods in Enzymology 82: 33-64. It can be purified by a method of
retaining the telopeptide (e.g., U.S. Pat. No. 3,114,593), or
alternatively, by a method of removing the telopeptide (e.g., U.S.
Pat. No. 4,233,360). It can also be reconstituted by cross-linking
using a chemical reagent (e.g., U.S. Pat. Nos. 5,876,444 and
6,177,514) or by other means (e.g., UV light). Collagen can be
covalently bonded to a hydroxyapatite-based matrix. The covalent
bond can be formed directly between a functional group in collagen
(e.g., carboxylate) and a functional group in modified
hydroxyapatite (e.g., amino), or formed indirectly through a third
molecule, e.g., a cross-linker. A cross-linker is an agent that has
two functional groups. One of them can form a bond with the
biopolymer and the other with the matrix. Examples of cross-linkers
include, but are not limited to, glutaraldehyde, tresyl chloride,
and N-hydroxysuccinumide.
[0010] Osteoprotegerin is an example of a bone formation promoter,
which may be deposited in the pores of the matrix described above.
Osteoprotegerin is a protein of the TNF receptor superfamily. It
has activities associated with bone metabolism, in particular, the
activity of inhibiting bone resorption thereby increasing bone
density. Simonet et al. (1997) Cell 89(2): 309-19. Rat
osteoprotegerin is a 401 amino acid protein, 85% and 94% homologous
to mouse and human osteoprotegerins, respectively. The term
"osteoprotegerin" herein refers to a polypeptide having a full or
partial amino acid sequence of the rat, mouse, or human
osteoprotegerin (see, for example, U.S. Pat. No. 6,015,938) or a
derivative thereof, and having the activity of inhibiting bone
resorption. When an implant is used for replacing a bone defect, it
is preferred that it contain a bone formation promoter in a
sufficient amount (e.g., 0.02% to 0.1% by weight) to promote bone
growth and inhibit bone resorption. The bone formation promoter may
be attached to the matrix via covalent bonding by methods well
known in the art.
[0011] A bone implant of this invention can be prepared as follows:
Porous hydroxyapatite is prepared by a hydrothermal process as
described in, e.g., Roy and Linnehan (1974) Nature 247: 220-222, or
by a process using organic particles as described in, e.g., Liu
(1996) Biomaterials 17: 1955-57; and Liu (1997) Ceramic
International 23: 135. During the preparation process, porous
hydroxyapatite is mold into a designed shape to obtain a
hydroxyapatite-based matrix. Then, the shaped matrix is immersed in
a solution containing a cross-linker, which has at least two
functional groups. One of the two functional groups reacts with the
matrix, and a covalent bond is formed between the cross-linker and
the matrix. Another solution containing a biopolymer, and
optionally, a bone formation promoter is prepared. In particular,
one can mix a biopolymer-containing solution with a bone formation
promoter-containing solution to obtain a homogenous solution. The
matrix having the cross-linker is then immersed in the
just-described solution for a sufficient period of time to form
another covalent bond between the cross-linker and the biopolymer
(and the bone formation promoter, if present) via the second
functional group in the cross-linker. The matrix is then removed
from the solution and lyophilized.
[0012] If a bone formation promoter is not included in the bone
implant thus obtained, it can be attached to the matrix by
immersing the bone implant in a solution containing such a
promoter, followed by air-drying or freeze-drying. By either
method, a bone formation promoter is deposited on both the external
and internal surfaces of the porous matrix.
[0013] A regenerative bone implant thus prepared can be used by
following standard surgical procedures to replace a bone
defect.
[0014] The specific examples below are to be construed as merely
illustrative, and not limitative of the remainder of the disclosure
in any way whatsoever. Without further elaboration, it is believed
that one skilled in the art can, based on the description herein,
utilize the present invention to its fullest extent. All
publications cited herein, including patents, are hereby
incorporated by reference in their entirety.
[0015] Preparation of Materials
[0016] Preparation of a porous hydroxyapatite-based matrix.
Hydroxyapatite powder is prepared by a wet chemical method
involving the reaction:
10Ca(NO.sub.3).sub.2+6(NH.sub.4).sub.3PO.sub.4+2NH.sub.3H.sub.2O=Ca.sub.1-
0(PO.sub.4).sub.6(OH).sub.2+20NH.sub.4NO.sub.3. A porous
hydroxyapatite-based matrix is prepared by the following steps: (i)
preparing a slurry, which includes hydroxyapatite powder, silicon
carbide, magnesia, and water; (ii) molding a network substrate
(e.g., polyurethane, polyvinyl chloride, or polyethyleneglycol)
into a desired shape; (iii) coating the slurry onto the network
substrate; and (iv) removing extra slurry by centrifugation. If
necessary, steps (i)-(iv) are repeated. The thus obtained
hydroxyapatite-containing substrate is sintered at a temperature of
1200.degree. C. and then cooled down. The temperature is increased
slowly so that the network substrate is decomposed gradually and no
cracks are formed. Thus, a porous hydroxyapatite-based matrix is
obtained, with an average pore size of 200-350 .mu.m. After
washing, the matrix is sterilized by gamma ray irradiation (20
kGy).
[0017] Extraction and purification of type I collagen. Type I
collagen is extracted and purified from tendons of New Zealand
white rabbits. The tendons are dissected, sliced, and washed with
several changes of cold distilled water to remove plasma proteins,
and then extracted by constant stirring overnight at 4.degree. C.
with 0.5 M NaCl in 50 mM Tris-HCl, pH 7.4. The supernant is
decanted and the reminder is washed with several changes of cold
distilled water to remove salts and then incubated overnight at
4.degree. C. with 0.5 M HOAc pH 2.5 to obtain an aqueous extract. A
salt solution (0.9 M NaCl) is added to the extract, causing
precipitation. The precipitation is collected by centrifugation at
13,000 rpm for 30 min, and dissolved in 0.05 M HOAc to form a
collagen-containing solution. Another salt solution (0.02 M
Na.sub.2HPO.sub.4) is added twice to the collagen-containing
solution over a 24 to 48 hr period causing precipitation. The
precipitation is collected by centrifugation, and dissolved in 50
mM HOAc to obtain another collagen-containing solution. The
collagen-containing solution is dialyzed against 5 mM HOAc, and
finally lyophilized.
[0018] Expression of recombinant osteoprotegerin. Constructing an
expression plasmid is well known in the art. See Simonet et al.
(1997) Cell 89(2): 309-19. For example, Human full-length (2.4 kb)
osteoprotegerin (OPG)-Fc fusion protein is constructed by PCR
amplification, and cloned into the plasmid vector pCEP4
(Invitrogen, San Diego, Calif.). The pCEP4OPG-Fc vector is then
lipofected into cells, e.g. 293-EBNA-1 cells (Invitrogen, San
Diego, Calif.), or Chinese Hamster Ovary cells using the
manufacturer's recommended methods. The OPG-Fc fusion protein is
expressed, and further purified by protein A/G-affinity
chromatography.
[0019] Preparation of a Bone Implant
[0020] Collagen (type I) is purified, digested with pepsin to
remove the telopeptide, and reconstituted by several steps of
modifications to form a glutaraldehyde-polymer amine complex (see,
e.g., U.S. Pat. No. 5,876,444). Both collagen and osteoprotegerin
are gamma ray sterilized and dissolved in 5 mM HOAc and a phosphate
buffer saline buffer, respectively. The collagen-containing
solution and the osteoprotegerin-containing solution are gently
mixed and heated to 30-40.degree. C. to facilitate mixing, if
necessary. A solution containing reconstituted collagen and
osteoprotegerin is obtained, and includes 0.2-1% by weight
osteoprotegerin, and 99-99.8% by weight collagen.
[0021] A porous hydroxyapatite-based matrix is prepared as
described above. An amino group is introduced to the surface of the
porous hydroxyapatite-based matrix by ammonia plasma. Then, the
matrix is immersed in a solution containing glutaraldehyde to form
a covalent bond between the amino group and glutaraldehyde. The
thus obtained matrix is further immersed in the above-described
solution containing reconstituted collagen and osteoprotegerin for
a sufficient period of time to form another covalent bound between
glutaraldehyde and the collagen and between glutaraldehyde and the
osteoprotegerin. Finally, the matrix is removed from the solution
and lyophilized to produce a bone implant. The pore sizes of the
collagen in the bone implant are in the range of 50 .mu.m to 200
.mu.m.
Other Embodiments
[0022] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated
otherwise, each feature disclosed is only an example of a generic
series of equivalent or similar features.
[0023] From the above description, one skilled in the art can
easily ascertain the essential characteristics of the present
invention, and without departing from the spirit and scope thereof,
can make various changes and modifications of the invention to
adapt it to various usages and conditions. Thus, other embodiments
are also within the claims.
* * * * *